Immunotherapeutical method for treating cancer

ABSTRACT

Pharmaceutical compositions useful as vaccines are described containing a purified surface or excreted protein qualitatively or quantitatively associated with a type of cancer, at least one interleukin (IL), and at least one colony stimulating factor (CSF), where the purified surface or excreted protein is provided in an amount sufficient to induce an immune response in an individual administered the composition. Such compositions can be used in methods for treating individuals having cancer, and for inducing an immunotherapeutic response in the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/005,993 having a filing date of Jan. 13, 2011, which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to methods and compositions comprisingpurified surface or excreted proteins for treating cancer.

BACKGROUND OF THE INVENTION

The National Cancer Institute estimates that over 10 million Americanshave a history of cancer. The American Cancer Society has predicted thatover 1.4 million new cancer diagnoses will be made and over half amillion Americans will die of cancer this year. Cancer is the secondmost common cause of death in the United States, exceeded only by heartdisease.

Given the prevalence of various forms of cancer in this country,significant research focuses on developing new methods of diagnosis andtreatment. Depending upon the type of cancer and its stage, currentforms of treatment typically can include surgery, radiation,chemotherapy and/or hormonal treatments.

In recent years, research efforts also have focused on the developmentof cancer vaccines. Such vaccines are intended either to treat existingcancers (i.e., therapeutic vaccines) or to prevent cancer fromdeveloping (i.e., prophylactic vaccines). Therapeutic vaccines aredesigned to treat cancers by stimulating the immune system to recognizeand attack cancer cells without damaging non-cancerous cells.Prophylactic vaccines are administered to healthy persons to stimulatetheir immune systems to attack cancer causing agents such as cancercausing viruses.

Currently, two vaccines have been licensed by the U.S. Food and DrugAdministration to prevent viral infections that can lead to cancer. Oneis a vaccine which prevents infection with the hepatitis B virus, avirus associated with some forms of liver cancer; the second is avaccine which prevents infection with two types of human papilloma virusthat together cause 70 percent of cervical cancer cases.

Progress in developing therapeutic cancer vaccines has come more slowly.Efforts have been made to vaccinate cancer patients with tumor cells ortumor-associated antigens. Some of these efforts have had limitedsuccess. An example of a class of vaccines is reported in U.S. Pat. No.5,478,556, which describes vaccinating breast cancer patients withcompositions comprising a combination of extracted autologous orallogeneic breast tumor associated antigens (TAA) obtained from cancercells, interleukin-2 (IL-2), and granulocyte macrophage colonystimulating factor (GM-CSF).

SUMMARY OF THE INVENTION

In one aspect, compositions are provided comprising (i) a purifiedsurface or excreted protein qualitatively or quantitatively associatedwith a type of cancer; (ii) at least one interleukin (IL); and (iii) atleast one colony stimulating factor (CSF), wherein the protein isprovided in an amount sufficient to induce an immune response in anindividual administered the composition.

In another aspect, methods for treating an individual are providedcomprising administering to an individual having a type of cancer, atherapeutically effective amount of a composition of the precedingaspect.

In another aspect, methods for inducing an immunotherapeutic response inan individual are provided comprising administering to an individualdiagnosed with a type of cancer a therapeutically effective amount of acomposition of the preceding aspect (supra), wherein the compositioncomprises the purified surface or excreted protein in an amountsufficient to induce an immunotherapeutic response in the individual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Kaplan-Meier Overall Survival Curve with patients who diedof other causes censured for Reference Example 1.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides methods and compositions useful for thetreatment of cancer. Thereby, in accordance with the present disclosure,a patient suffering from a type of cancer is administered atherapeutically effective amount of a composition comprising a purifiedsurface or excreted protein which is qualitatively or quantitativelyassociated with that cancer type in combination with adjuvantscomprising an interleukin (IL) and a colony stimulating factor (CSF).

The disclosure further is directed to cancer vaccine compositionscomprising a purified surface or excreted protein qualitatively orquantitatively associated with particular types of cancer in combinationwith biological adjuvants comprising an interleukin and a colonystimulating factor.

Without being limited by any one mechanism of action, it is believedthat the composition acts by causing tumor cell death, inhibiting tumorgrowth or recurrence, promoting tumor regression and/or inhibition ofmetastasis, leading to an increase in disease-free survival rate and/oroverall survival rate; or any combination thereof.

In certain embodiments, the interleukin is interleukin-2 (IL-2) and thecolony stimulating factor is granulocyte-macrophage CSF (GM-CSF).

The immune response of a patient receiving the vaccine can be measuredprior to the first vaccination and then measured again after the firstvaccination and, if desired, after any subsequent vaccination todetermine the extent of the immune response. The level of immuneresponse can be quantified with the Lymphocyte Blastogenesis Assay (LBA)(see Elliott, R. et al., Breast Cancer Research and Treatment 30:299-304(1994); herein incorporated by reference). In this assay, mononuclearcells are grown in tissue culture for seven days. The mononuclear cellsare co-cultured with tumor antigen in varying concentrations.³H-thymidine is added to the cultures shortly before termination. Aproliferation index is then calculated by dividing the amount of³H-thymidine uptake incorporated into tumor antigen-stimulatedmononuclear cells in culture by the ³H-thymidine uptake incorporatedinto control mononuclear cells, which were not co-cultured with tumorantigen.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of the referenced compound or composition that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician, which includes one or more of thefollowing:

-   (1) preventing the disease; for example, preventing a disease,    condition or disorder in an individual who may be predisposed to the    disease, condition or disorder but does not yet experience or    display the pathology or symptomatology of the disease;-   (2) inhibiting the disease; for example, inhibiting a disease,    condition or disorder in an individual who is experiencing or    displaying the pathology or symptomatology of the disease, condition    or disorder; and-   (3) ameliorating the disease; for example, ameliorating a disease,    condition or disorder in an individual who is experiencing or    displaying the pathology or symptomatology of the disease, condition    or disorder (i.e., reversing the pathology and/or symptomatology)    such as decreasing the severity of disease.    For example, a therapeutically effective amount of a compound or    composition can be measured as an amount that increases the immune    response to tumor antigens by at least 50%; or stabilizes or    decreases circulating tumor marker proteins resulting in inhibiting    the progression of the cancer.

As used here, the terms “treatment” and “treating” means (i)ameliorating, inhibiting, or preventing the referenced disease state, asdescribed above; or (ii) eliciting the referenced biological effect(e.g., an immunotherapeutic reaction can result in an alleviation orarrest of the disease from which the patient of interest is suffering).More specifically, treating can include inhibiting tumor growth orrecurrence, regressing a tumor and/or inhibiting formation ofmetastases.

As used herein, reference to “a” or “an” or “the” protein or antigen oradjuvant can refer to one or more than one protein or antigen oradjuvant. As used herein, a surface or excreted protein which is“qualitatively or quantitatively associated with” a cancer type means aprotein which is either typically found only on the surface of, orexcreted by, one or more particular types of cancer or is found on thesurface of, or excreted by, cancer cells more abundantly than normal ornon-cancerous cells. Such proteins also are known as tumor markerproteins or tumor associated antigens, and these terms are usedinterchangeably within the application. These proteins can be purifiedfrom a tumor cell preparation or can be synthesized by methods known topersons of skill in the art. Further, these proteins can be prepared asa mixture with other non-tumor associated proteins, or can be preparedin a preparation where they are the only protein component.

Examples of such proteins or antigens, and the cancers with which theyare associated, include those listed in Table 1 below:

TABLE 1 Cancer Proteins & Antigens prostate PSA, prostatic acidphosphatase (PAP), prostate specific membrane antigen (PSMA),carcino-embryonic antigen (CEA), cancer antigen (CA) 125 breast CA 15-3,CA 27.29, CEA, CA 125, estrogen receptor (ER), progesterone receptor(PgR), human epidermal growth factor receptor 2 (Her-2/neu),lipid-associated sialic acid in plasma (LASA-P) gastroin- CEA, CA 19-9,α-feto protein (AFP), CA 72-4, bladder testinal tumor antigen (BTA),tissue polypeptide antigen (TPA) lung CEA, AFP, neuron-specific enolase(NSE), chromogranin A, TPA, CA 72-4 liver CEA, CA 19-9, AFP, CA 50pancreatic CEA, CA 19-9, AFP thyroid CEA, calcitonin, thyroglobulinovarian CA 125, AFP, LASA-P, CA 50, CA 72-4 melanoma S-100 proteins(S-100), tumor-associated antigen 90 (TA-90) testicular AFP, β Humanchorionic gonadotropin (β-hCG); leukemia β-2 microglobulin, LASA-Pcolorectal CEA, CA 19-9, LASA-P bladder BTA, nuclear matrix protein 22(NMP 22), TPA lymphoma: LASA-P endometrial ER, PgR nervous calcitonin,chromogranin A, NSE system embryonic β-hCG

The compositions can be made from isolated and purified autologous,allogeneic or recombinant surface or excreted tumor marker proteins, ora combination thereof. These compositions have been found to be usefulas cancer vaccines when administered in combination with low doses of aninterleukin and a CSF.

In one embodiment, the composition comprises IL-2 and GM-CSF.

The use of isolated and purified antigens to the particular type ofcancer to be treated provides more specificity and eliminates thevariability present in the prior art vaccine compositions. Furthermore,preparation of the present vaccine does not require tissue from thepatient and therefore every patient at presentation or after surgery isa candidate for the therapeutic vaccine. However, if desired, tissuefrom a patient can be used to make the present vaccine. The use ofisolated and purified antigens also allows the vaccine compositions tobe mass produced.

As used herein, the term “purified” means that the referenced entity hasa purity of greater than about 50% by total weight. That is, in certainembodiments, each of the isolated and purified antigens used herein,independently, have a purity of greater than about 50% by total weight;or greater than about 60% by weight; or about 70% by weight; or about80% by weight; or about 85% by weight; or about 90% by weight; or about95% by weight; or about 98% by weight; or about 99% by weight.

The term “about” as used herein means+/−10% of the referenced value.

The compositions can be administered to patients suffering from solidtumors, malignant ascites or hemopoietic cancers to inhibit tumor growthor recurrence or to inhibit formation of metastases. The compositionsalso can be administered to patients suffering from leukemia orlymphoma.

Vaccine compositions are made using tumor marker proteins. Desiredisolated and purified tumor marker proteins are available commercially.Sources of many of the antigens listed above include FitzgeraldIndustries International, Concord, Mass., and Sigma-Aldrich Co, Dallas,Tex. Other commercial sources are known and available to persons ofskill in the art. If any particular antigen is not commerciallyavailable, it can be synthesized in a cGMP facility according to methodsknown by persons of skill in the art.

Each of the tumor marker protein(s) are independently present in amountstypically ranging from about 1 μg to about 1000 μg per 100 μL of vaccinecomposition, or about 1 μg to about 250 μg per 100 μL of composition, orabout 25 to about 100 μg per 100 μL of composition, or about 50 μg per100 μL of composition, or about 1 μg to about 10 μg per 100 μL. If aparticular antigen is not readily available or is not readily availableat a reasonable cost, smaller amounts within the preceding ranges, suchas within the range of about 1 μg to about 10 μg per 100 μL, can be usedwith good effect.

Some tumor marker proteins are sold on the basis of international units(IU), rather than μg. In such an instance, the antigen typically isprovided in an amount within the range of 1 IU to about 10,000 IU per100 μL of vaccine composition; or about 20 to about 5,000 IU, per 100 μLof composition; or about 500 IU to about 1500 IU per 100 μL ofcomposition, or about 1000 IU per 1000 μL of composition.

For example, if the type of cancer of interest is prostate cancer andPSA is included in the vaccine composition, PSA can be provided at aconcentration of about 1 μg to about 250 μg per 100 μL of composition,or about 25 μg to about 100 μg per 100 μL of composition, or about 50 μgper 100 μL of composition. PSA can be the sole marker in the prostatecancer vaccine, or the vaccine can comprise one or more additional oralternative antigens as indicated above.

The vaccine, for example, can also comprise CEA; this antigen typicallyis provided at a concentration of about 1 μg to about 250 μg per 100 μLof composition. CA 125 protein can be included in the vaccine; ittypically is sold in the form of IU and can be provided at aconcentration of from about 20 IU to about 5,000 IU per 100 μL ofcomposition, or from about 100 IU to about 2500 IU per 100 μL ofcomposition, or about 1000 IU, per 100 μL of composition.

CA 15-3 and/or CA 27.29 can be included, each at a concentration ofabout 1 μg to about 250 μg per 100 μL of composition.

Generally, the amount of a particular antigen used in the vaccineremains about the same whether the antigen is the sole antigen providedin the vaccine or is one of a number of antigens provided in the vaccinecomposition. Thus, in the prostate cancer vaccine example provided inthe preceding paragraph, whether the composition comprises one of theantigens indicated or a combination of two, three, four or fiveantigens, each antigen is provided in an amount within the guidelinesset forth above. The presence of additional antigens can increase theprobability of an effective immune response in the patient.

In another embodiment, the purified antigen(s) are physically combinedwith small amounts of a combination of an interleukin and CSF. Inanother embodiment, the antigen solution and the adjuvants (e.g, theinterleukin and CSF) are administered separately, but the antigens andadjuvants should be administered to the same site as close together intime as possible and no more than 2 hours apart. Typically, eachcomponent of the vaccine is provided in the form of a physiologicallyacceptable solution and then desired amounts of each solution arecombined to make the final vaccine composition.

Suitable interleukins include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-9, IL-12, IL-13, IL-14 and IL-15. In certain embodiments, theinterleukin is IL-1. In certain other embodiments, the interleukin isIL-2. In certain other embodiments, the interleukin is IL-3. In certainother embodiments, the interleukin is IL-4. In certain otherembodiments, the interleukin is IL-5. In certain other embodiments, theinterleukin is IL-6. In certain other embodiments, the interleukin isIL-7. In certain other embodiments, the interleukin is IL-9. In certainother embodiments, the interleukin is IL-12. In certain otherembodiments, the interleukin is IL-13. In certain other embodiments, theinterleukin is IL-14. In certain other embodiments, the interleukin isIL-15.

Suitable colony stimulating factors include granulocyte-macrophage-CSF(GM-CSF), granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF). In certainembodiments, the colony stimulating factor is GM-CSF. In certainembodiments, the colony stimulating factor is G-CSF. In certainembodiments, the colony stimulating factor is M-CSF.

A single interleukin and a single colony stimulating factor can be usedin the compositions herein or a combination of two or more interleukinsand/or colony stimulating factors can be used. Although theeffectiveness of each type of cytokine is very limited if administeredalone, the administration of both an interleukin and a CSF incombination with the tumor marker antigens has been found to have asynergistic effect.

In one embodiment, a combination of cytokines is GM-CSF and IL-2. TheIL-2 promotes cytotoxic T-cell immunity; the GM-CSF promotes dendriticcell processing.

Typically, the composition comprises from about 5,000 U to about 50,000Units (U) (about 0.3 μg to about 3 μg) of the interleukin per 100 μL ofcomposition; or from about 10,000 U to about 30,000 U (about 0.6 μg toabout 1.8 μg) of the interleukin per 100 μL of composition; or about20,000 U (about 1.2 μg) of the interleukin per 100 μL of composition andabout 10 μg to about 100 μg of the CSF per 100 μL of composition, orabout 15 to about 20 μg of the CSF per 100 μL of composition, or about16.5 μg to about 16.9 μg of the CSF per 100 μL of composition, or about16.7 μg of the CSF per 100 μL of composition.

Typically, each cytokine is supplied as a lyophilized powder andcombined with a physiologically acceptable liquid carrier and seriallydiluted if necessary to reach the final desired concentration in 100 μL.IL-2, for example, can be obtained under the name Proleukin® from ChironCorporation, Emeryville, Calif., as a lyophilized powder, 22×10⁶ IU (1.3mg)/vial. If, for example, it is combined with 1.1 mL of sterile waterand then serially diluted 1:10, 1:10 (final 1:100), its finalconcentration is 20,000 IU (1.18 μg) in 100 μL. Similarly, GM-CSF can beobtained under the name Leukine® from Berlex Laboratories, Montville,N.J., as a lyophilized powder, 250 μg/vial. If, for example, it iscombined with 1.5 mL of sterile water, a final concentration of 16.7 μgper 100 μL will be obtained.

Suitable pharmaceutically acceptable carriers for the compositionsherein refer to fluid vehicles that can be injected into a host withoutsignificant adverse effects. Suitable pharmaceutically acceptablecarriers known in the art include, but are not limited to, sterilewater, saline, glucose, and physiologically acceptable aqueous buffersor solutions, including saline or phosphate-buffered saline. Carriersmay include auxiliary agents including, but not limited to, diluents,stabilizers (i.e., sugars and amino acids), preservatives, wettingagents, emulsifying agents, pH buffering agents, viscosity enhancingadditives, colors and the like. For example, suitable carriers for thetumor marker proteins and the adjuvants include physiologicallyacceptable aqueous buffers or solutions, including sterile water,saline, phosphate-buffered saline, or Hank's buffered salt solution(HBSS).

Appropriate amounts of the antigens or adjuvants are mixed with theselected carrier. All of the components of the vaccine can be providedtogether in one carrier, but if desired, one or more components can beprovided in a separate carrier and administered in combination with theother components.

In another aspect the disclosure provides methods for treating cancerwhich comprises administering a therapeutically effective amount of acomposition comprising a purified surface or excreted protein which isqualitatively or quantitatively associated with the cancer type incombination with biological adjuvants comprising an interleukin (IL) anda colony stimulating factor (CSF).

The vaccine can be administered by injection. In another embodiment, thevaccine is administered subcuticularly, but it also can be administeredintramuscularly, subcutaneously, or intraperitoneal. The vaccinetypically is administered in doses of about 0.3 mL to about 1 mL, orabout 0.5 mL to about 0.7 mL, or about 0.5 mL, per dose. Although asingle dose of the vaccine may be useful, often times it is desirable toadminister from about 3 to about 12 doses of the vaccine and or about 6to about 12 doses. If more than one dose is administered, the dosestypically are at least 7 days apart. For example, the doses typicallycan be administered about 7 to about 90 days apart. For example, oneuseful regimen is to administer three doses of the vaccine at weeklyintervals, then to administer a further three doses at four weekintervals after that (i.e., the vaccine is administered at weeks 0, 1,2, 6, 10 and 14). If desired, booster shots can be administered monthlyor bi-monthly for up to about one year following the initialimmunization therapy. The booster shots can be alternated with boostershots of interleukin, such as, but not limited to, IL-2, to boost thepatient's natural killer cells.

The T- and B-cell immunity of a patient receiving a vaccine inaccordance with this disclosure can be monitored using a lymphocyteblastogenesis assay (see Elliott, R. et al., Breast Cancer Research andTreatment 30:299-304 (1994); herein incorporated by reference) beforebeginning the vaccine administration and then during and after thevaccine regimen. An increase in the proliferation index demonstrates anincrease in the immune response to the cellular and protein antigens bythe vaccination process. A patient receiving a vaccine in accordancewith this disclosure can also be monitored with an assay measuringcirculating tumor marker proteins. A decrease in circulating tumormarker proteins is a surrogate marker for an increase in the immuneresponse.

The vaccines herein are useful for the treatment of any solid cancer,including cancers of the prostate, breast, lung, colon, rectum, uterus(including cervix and endometrium), ovary, oral cavity, bladder,pancreas, stomach, liver, kidney, skin, testicles and lymphoid tissue.They also can be used to treat malignant ascites and hematopoietictumors, including leukemias and lymphomas. Appropriate tumor markerproteins are selected, and the vaccine is prepared using the generalamounts of the components of the vaccine as set forth above.

In one embodiment, compositions are provided that are useful as aprostate cancer vaccine which comprise at least one purified surface orexcreted protein qualitatively or quantitatively associated withprostate cancer combined with a combination of biological adjuvants,wherein the adjuvants consist essentially of an interleukin and a colonystimulating factor. In one embodiment, at least one purified surface orexcreted protein comprises purified PSA, optionally in combination withone or more other purified proteins associated with prostate cancer. Ina related embodiment, a method is provided for treating a person withprostate cancer which comprises immunizing the person with a compositioncomprising purified PSA, optionally in combination with one or moreother purified proteins associated with prostate cancer, and biologicaladjuvants comprising an interleukin and a colony stimulating factor. Ina further related embodiment, a method is provided for eliciting in ahuman with prostate cancer an immunotherapeutic response, wherein theimmunotherapeutic response results in a decrease in circulating PSA anddeath to tumor cells, by immunizing the person with a compositioncomprising purified PSA, optionally in combination with other purifiedproteins associated with prostate cancer, and biological adjuvants,wherein the adjuvants consist essentially of an interleukin and a colonystimulating factor. The immunizations can be carried out once or, can berepeated. The immunization is designed to increase the immunotherapeuticresponse to the tumor antigens/proteins by a minimum of about 50% overbaseline immunity, decrease circulating PSA, and result in tumor celldeath, increased progression free survival and overall survival.

Another embodiment includes compositions useful as a breast cancervaccine, comprising therapeutically effective amounts of purifiedsurface or excreted proteins qualitatively or quantitatively associatedwith cancer of the breast. Such compositions can include CA 15-3, CA 125or CEA, or a combination thereof, together with the above combination ofbiological adjuvants. In a related embodiment, a method is provided fortreating a person with breast cancer which comprises immunizing theperson with a composition comprising purified CA 15-3, CA 125 and CEA,optionally in combination with one or more other purified proteinsassociated with breast cancer, and biological adjuvants comprising aninterleukin and a colony stimulating factor. The immunizations can becarried out once or can be repeated. The immunization is designed toincrease the immunotherapeutic response to the tumor antigens/proteinsby a minimum of about 50% over baseline immunity, decrease circulatingtumor antigens, and result in tumor cell death, increased progressionfree survival and overall survival.

Other embodiments include compositions comprising therapeuticallyeffective amounts of purified proteins qualitatively or quantitativelyassociated with cancer of the gastrointestinal system, lung, liver,pancreas, thyroid, ovaries, testicles, colorectal system, endometrium,bladder or nervous system, or melanoma, leukemia or lymphoma, and theadministration of these compositions to persons suffering from suchcancer to elicit an immune response to the cancer.

The disclosure is further illustrated by the following examples, whichare not intended to be limiting.

EXAMPLES Reference Example 1

A retrospective study was performed to determine the relationshipbetween immune response and overall survival. Immune response wasmeasured with the LBA, as discussed above.

The study was performed on early stage breast cancer patients withdepressed immunity who had received a breast cancer vaccine in theadjuvant setting. The patients were tested for immune responses toautologous cells, allogeneic cells and protein antigens. Patients withdepressed immunity, a ratio less than 1.5 (determined by cut pointanalysis of data set (Head, et al., Ann. NY Acad. Sci, 1993; 690:340-342) of present modified LBA assay) in the LBA, were vaccinated witha minimum of six vaccines containing autologous breast cancer cells,allogeneic breast cancer cells (MCF-7 cells), CA 15-3 antigen, CA 125antigen, CEA antigen and biological adjuvants (IL-2 and GM-CSF). Asecond LBA was done 2 to 4 weeks after the 6^(th) vaccination. Thefollow-up has been for up to 10 years. FIG. 1 is the Kaplan-MeierOverall Survival Curve with patients who died of other causes censured.The calculated Kaplan Meier overall survival (i.e., the percent survivalover a continuous time period) for the patients at 10 years is 59% indepressed patients with standard treatment, and 95% survival forpatients with immunity to their own tumor associated antigens in a LBAat presentation with standard treatment (historic controls for study).Depressed immunity patients lacking immunity to their own tumor antigensat presentation were vaccinated as stated above and this lead to anincrease in overall survival from 59% to 79% at 10 years. The groupswere well matched for age, menopausal status, tumor size, nodal status,stage of disease, estrogen receptor status, progesterone receptor statusand antihormone therapy

This study shows that patients with depressed immunity have a loweroverall survival and a poor long-term prognosis when compared topatients with normal immunity, and further, that improving the immuneresponse through vaccination improves overall survival.

Further, it is generally known in the art that an increase in immuneresponse correlates to increase in overall survival. For instance, Hsuehet al. (J. Clin. Oncol., Dec. 1, 2002; 20(23): 4549-54), incorporatedherein by reference, states “[s]urvival after vaccine immunotherapy wassignificantly correlated with the DTH immune response to vaccine but notto a control antigen . . . ” (p. 4553, first paragraph of Discussion).In addition, Galon et al. (Science, Sep. 29, 2006; 313: 1960-64),incorporated herein by reference, reported that the “ . . . time torecurrence and overall survival time are governed in large part by thestate of the local adaptive immune response” (p. 1963, last paragraph).

Therefore, it is both known in the art and demonstrated by the data inFIG. 1, that an improved immune response correlates with overallsurvival in cancer patients.

Example 1 Preparation of PSA Composition

A composition in accordance with the present disclosure was made inaccordance with the following procedure:

1. One mg of PSA (Fitzgerald Industries International, Concord, Mass.,CAT #30-AP15E) was diluted in 2.0 mL of sterile water USP. The solutionwas filter sterilized with a 0.22 micron filter. 100 μL sterile aliquotswere frozen at −80° C. until needed.

2. 500 μg of CEA (Fitzgerald Industries International, Concord, Mass.,CAT #30-AC30) were diluted in 25 mL sterile water USP and the resultantsolution was filter sterilized with a 0.22 micron filter. 100 μL sterilealiquots were frozen at −80° C. until needed.

3. 20,000 units of CA 125 (Fitzgerald Industries International, Concord,Mass., CAT #30-AC11) were diluted in 2.0 mL sterile water USP and theresultant solution was filter sterilized with a 0.22 micron filter. 100μL sterile aliquots were frozen at −80° C. until needed.

4. 22 million units of IL-2 (Proleukin; Chiron Corporation, Emeryville,Calif.) were reconstituted in 1.1 mL sterile water USP, then diluted1:10 with sterile water USP. The resultant solution was diluted 1:10again to provide a final concentration of 20,000 units per 100 μL.Sterile aliquots of 100 μL were frozen at −80° C. until needed.

5. A vial of 250 μg GM-CSF (Berlex Laboratories, Montville, N.J.) wasreconstituted with 1.5 mL sterile water USP to provide a finalconcentration of 16.7 μg in 100 μL. The solution was refrigerated at 4°C.

6. To prepare a dose of the vaccine, frozen aliquots of the followingwere brought to room temperature: 100 μL of each of the three antigens(PSA, CEA and CA 125) and 100 μL of each of the two adjuvants (IL-2 andGM-CSF). Then 100 μL of each component were drawn into a 1 mL syringewith a one inch 26 gauge needle. The final total volume was 0.5 mL. Eachdose of vaccine contained 50 μg PSA; 2 μg CEA; 1000 IU CA 125; 20,000units IL-2; and 16.7 μg GM-CSF.

Example 2

Thirteen biopsy-confirmed prostate cancer patients were administeredsubcuticularly at 0, 1, 2, 6, 10 and 14 weeks an initial course of 6vaccinations, each dose containing PSA, CEA, CA 125, IL-2 and GM-CSF,prepared as in Example 1. Each patient's serum PSA level was determinedbefore the first vaccine was initiated. During the vaccination periodthe patients received no other concurrent therapy (i.e., surgery,hormones, radiation, radioactive seeds or chemotherapy).

The treatment protocol was designed according to the scheme taught byGehan (see Gehan, E., J. Chron. Dis. 13(4), 346-353 (1961); incorporatedherein by reference) for determining the minimum response rates foroncology drugs in single arm Phase I/II clinical trials. Briefly,Gehan's method calculated the minimum sample size required without anyresponses to terminate the study because a predetermined minimumresponse rate (typically 15 to 20%) will not be reached. The confidencelevel was set at 95% and thus the study would be terminated early ifthere was 95% confidence that the response rate was less than the targetresponse rate of 15 to 20%. Therefore, if 14 patients for 20% responserate or 19 patients for 15% response rate are treated without anyresponses then the drug is inactive. If one patient responds, then morepatients need to be treated to determine an accurate response rate.

The results of the administration of the prostate cancer vaccine areprovided in Table 2 below:

TABLE 2 PSA PSA AFTER PSA AFTER BEFORE 6 12 LAST PSA DIAGNOSIS VACCINEVACCINES VACCINES (months) Prostate Cancer 4.10 2.40 2.50 3.50 (80)Prostate Cancer 1.40 0.60 0.66 0.90 (92) Prostate Cancer 6.80 6.40 only6 — vaccines Prostate Cancer 4.90 2.80 2.40 2.97 (42) Prostate Cancer6.20 5.80 1.90 2.20 (65) Prostate Cancer 4.20 3.50 4.40 3.90 (18)Prostate Cancer 14.60 5.50 6.50 7.70 (49) Prostate Cancer 7.6 13.70 only4 — vaccines Prostate Cancer 4.00 4.93 seed — implants Prostate Cancer8.95 10.60 17.19  Zoladex, seeds Prostate Cancer 7.20 5.41 7.30 6.00(28) Prostate Cancer 4.55 7.02 4.17 10.80 (21)  Prostate Cancer 5.4010.40 Lupron —The PSA level of patient #8 rose from 7.6 to 13.7 after the fourthvaccination and he withdrew from the study to seek other therapy. Theserum PSA level of each of the remaining 12 prostate cancer patients wasdetermined 3-4 weeks after the 6^(th) vaccination. As shown in Table 2,there was a decrease in serum PSA level in 8 of the 12 patients afterthe 6^(th) vaccination.

One patient (patient #3), whose PSA had dropped from 6.8 to 6.4 andpreviously had received radiotherapy (the only patient previouslytreated), elected to withdraw from the study at the end of this initialvaccination period and underwent a radical prostatectomy. Two patientswith rising PSAs after 6 vaccines elected to have additional standardtherapy (one patient received seed implants and the other Lupron). Nineof the original 13 patients received 3 further doses of the vaccinealternated with doses of IL-2 (11 million units) to boost their naturalkiller cells for the 6 months following the initial vaccinations. One ofthese patients with a rising PSA elected to take Zoladex and seedimplants. Eight patients have been followed for 18-92 months (variationin follow-up is due to the initial vaccination date of each patient (thepatients entered into the trial at different times) and patients beinglost to follow-up). The mean PSA value for these 8 patients wasinitially 5.9; it decreased to 4.1 after the initial 6 vaccines,decreased further to 3.7 after the 12 vaccines and was 4.7 after a meanfollow-up time of 49 months (median of 45.5 months).

One patient, whose PSA decreased from 4.1 to a normal 2.4 ng/ml aftervaccination, was re-biopsied after 18 and 30 months without cancer beingfound and has remained disease free and stable for over 10 years. Twoother patients also have been re-biopsied subsequent to the vaccinationprocess: one had no disease in the biopsy sample at 24 months and theother had a very significant decrease in the percentage of the biopsysample containing cancer cells (from 55% to 1%) at 16 months.

The prostate cancer vaccine, described above, had immunologicalresponses (as demonstrated by decreased serum PSA in 8 of 12 (67%)patients after six vaccines and 6 of 9 (67%) patients after 12 vaccines.Of the original 13 patients who enrolled in the trial 7 (54%) patientshad PSA levels lower than at presentation after a median of 45.5 months(range of 18 to 80 months) of follow-up which is indicative of nobiochemical progression of their disease (no increase in the amount ofserum PSA). These response rates, as measured by decreases in thebiological tumor marker PSA serum concentration, are estimated at 50% orbetter and therefore reflect a very significant response rate, accordingto the Gehan protocol. There is a slight increase in many of theprostate cancer patients PSA over time, and this suggests the need forbooster vaccines after the initial 12 vaccines.

Example 3

The vaccines provided herein also are used to treat patients with otherforms of cancer. Vaccine compositions are made using the followingcomponents following the general procedure in Example 1, as shown inTable 3.

TABLE 3 Cancer Marker 1 Marker 2 Marker 3 IL CSF breast 50 μg 50 μg 1000IU 20,000 16.7 μg CEA CA 15-3 CA 125 units GM-CSF IL-2 gastroin- 50 μg50 μg 50 μg alpha 20,000 16.7 μg testinal CEA CA 19-9 Fetoprotein unitsGM-CSF IL-2 liver 50 μg 50 μg 50 μg alpha 20,000 16.7 μg CEA CA 19-9Fetoprotein units GM-CSF IL-2 pancreatic 50 μg 50 μg 50 μg alpha 20,00016.7 μg CEA CA 19-9 Fetoprotein units GM-CSF IL-2 ovarian 1000 IU 50 μgalpha 20,000 16.7 μg CA 125 Fetoprotein units GM-CSF IL-2 colorectal 50μg 50 μg 20,000 16.7 μg CEA CA 19-9 units GM-CSF IL-2

We claim:
 1. A method for treating an individual comprisingadministering to an individual having a type of cancer, atherapeutically effective amount of a composition comprising about 50 μgPSA, about 2 μg CEA, about 1000 IU CA 125, about 20,000 units IL-2, andabout 16.7 μg GM-CSF, per dose.
 2. A method for inducing animmunotherapeutic response in an individual comprising administering toan individual diagnosed with a type of cancer a therapeuticallyeffective amount of a composition comprising about 50 μg PSA, about 2 μgCEA, about 1000 IU CA 125, about 20,000 units IL-2, and about 16.7 μgGM-CSF, per dose, and wherein the type of the cancer is prostate cancer.